Regulation Of Translation Elongation Research Articles

The DEAD-Box RNA Helicase Ded1 Is Associated with Translating Ribosomes.

DEAD-box RNA helicases are ATP-dependent RNA binding proteins and RNA-dependent ATPases that possess weak, nonprocessive unwinding activity in vitro, but they can form long-lived complexes on RNAs when the ATPase activity is inhibited. Ded1 is a yeast DEAD-box protein, the functional ortholog of mammalian DDX3, that is considered important for the scanning efficiency of the 48S pre-initiation complex ribosomes to the AUG start codon. We used a modified PAR-CLIP technique, which we call quicktime PAR-CLIP (qtPAR-CLIP), to crosslink Ded1 to 4-thiouridine-incorporated RNAs in vivo using UV light centered at 365 nm. The irradiation conditions are largely benign to the yeast cells and to Ded1, and we are able to obtain a high efficiency of crosslinking under physiological conditions. We find that Ded1 forms crosslinks on the open reading frames of many different mRNAs, but it forms the most extensive interactions on relatively few mRNAs, and particularly on mRNAs encoding certain ribosomal proteins and translation factors. Under glucose-depletion conditions, the crosslinking pattern shifts to mRNAs encoding metabolic and stress-related proteins, which reflects the altered translation. These data are consistent with Ded1 functioning in the regulation of translation elongation, perhaps by pausing or stabilizing the ribosomes through its ATP-dependent binding.

Disome-seq reveals widespread ribosome collisions that promote cotranslational protein folding

BackgroundThe folding of proteins is challenging in the highly crowded and sticky environment of a cell. Regulation of translation elongation may play a crucial role in ensuring the correct folding of proteins. Much of our knowledge regarding translation elongation comes from the sequencing of mRNA fragments protected by single ribosomes by ribo-seq. However, larger protected mRNA fragments have been observed, suggesting the existence of an alternative and previously hidden layer of regulation.ResultsIn this study, we performed disome-seq to sequence mRNA fragments protected by two stacked ribosomes, a product of translational pauses during which the 5′-elongating ribosome collides with the 3′-paused one. We detected widespread ribosome collisions that are related to slow ribosome release when stop codons are at the A-site, slow peptide bond formation from proline, glycine, asparagine, and cysteine when they are at the P-site, and slow leaving of polylysine from the exit tunnel of ribosomes. The structure of disomes obtained by cryo-electron microscopy suggests a different conformation from the substrate of the ribosome-associated protein quality control pathway. Collisions occurred more frequently in the gap regions between α-helices, where a translational pause can prevent the folding interference from the downstream peptides. Paused or collided ribosomes are associated with specific chaperones, which can aid in the cotranslational folding of the nascent peptides.ConclusionsTherefore, cells use regulated ribosome collisions to ensure protein homeostasis.

Genetic removal of eIF2α kinase PERK in mice enables hippocampal L-LTP independent of mTORC1 activity.

Characterization of the molecular signaling pathways underlying protein synthesis-dependent forms of synaptic plasticity, such as late long-term potentiation (L-LTP), can provide insights not only into memory expression/maintenance under physiological conditions but also potential mechanisms associated with the pathogenesis of memory disorders. Here, we report in mice that L-LTP failure induced by the mammalian (mechanistic) target of rapamycin complex 1 (mTORC1) inhibitor rapamycin is reversed by brain-specific genetic deletion of PKR-like ER kinase, PERK (PERK KO), a kinase for eukaryotic initiation factor 2α (eIF2α). In contrast, genetic removal of general control non-derepressible-2, GCN2 (GCN2 KO), another eIF2α kinase, or treatment of hippocampal slices with the PERK inhibitor GSK2606414, does not rescue rapamycin-induced L-LTP failure, suggesting mechanisms independent of eIF2α phosphorylation. Moreover, we demonstrate that phosphorylation of eukaryotic elongation factor 2 (eEF2) is significantly decreased in PERK KO mice but unaltered in GCN2 KO mice or slices treated with the PERK inhibitor. Reduction in eEF2 phosphorylation results in increased general protein synthesis, and thus could contribute to the mTORC1-independent L-LTP in PERK KO mice. We further performed experiments on mutant mice with genetic removal of eEF2K (eEF2K KO), the only known kinase for eEF2, and found that L-LTP in eEF2K KO mice is insensitive to rapamycin. These data, for the first time, connect reduction in PERK activity with the regulation of translation elongation in enabling L-LTP independent of mTORC1. Thus, our findings indicate previously unrecognized levels of complexity in the regulation of protein synthesis-dependent synaptic plasticity. Read the Editorial Highlight for this article on page 119. Cover Image for this issue: doi: 10.1111/jnc.14185.

Issue Cover (July 2018)

Front cover: Background: Long-lasting forms of memory and synaptic plasticity such as late long-term potentiation (L-LTP) are dependent on protein synthesis. The aim of our study is to investigate potential interactions between signaling pathways controlling protein synthesis in hippocampal L-LTP. Main findings: We found in mice that L-LTP failure caused by inhibition of the mammalian target of rapamycin complex 1 (mTORC1) is prevented by genetic deletion of PERK, a kinase for eukaryotic initiation factor 2α (eIF2α). A key mechanism responsible for the L-LTP rescue may involve down-regulation of translational factor eEF2 phosphorylation, which is associated with increased protein synthesis capacity via its upregulation on mRNA translation elongation. Implication: These data, for the first time, connect reduction of PERK activity with the regulation of translation elongation in enabling L-LTP independent of mTORC1. Image content: This image (63x) taken by confocal laser scanning microscope shows area CA1 of an acute hippocampal slice from wild-type mouse that was labeled for phospho-eEF2 (green) and DAPI (blue). Phospho-eEF2 is distributed in both soma and dendrites. Read the full article ‘Genetic removal of eIF2α kinase PERK in mice enables hippocampal L-LTP independent of mTORC1 activity’ by H. R. Zimmermann, W. Yang, B. C. Beckelman, N. P. Kasica, X. Zhou, L. D. Galli, A. G. Ryazanov, T. Ma (J. Neurochem. 2018, vol. 146 (2), pp. 133–144) on doi: 10.1111/jnc.14306

Widespread regulation of translation by elongation pausing in heat shock

Global repression of protein synthesis is a hallmark of the cellular stress response and has been attributed primarily to inhibition of translation initiation. Heat shocked cells globally inhibit protein synthesis, however the extent of translation initiation inhibition cannot explain the degree of translational repression observed.Exploring translational control during heat stress in mammalian cells using ribosome footprinting, we discovered that severe heat shock triggers elongation pausing of ribosomes after translating ~70 codons on the vast majority of mRNAs. The genome‐wide nature of the phenomenon, its location and the features of protein N‐termini suggested the involvement of ribosome‐associated chaperones.Reduced association of Hsp70 chaperones with ribosomes was seen following severe heat shock. Pre‐treatment with mild heat stress or overexpression of Hsp70 protected cells from the elongation pause induced by severe heat shock, while inhibition of Hsp70 activity triggered elongation pausing without heat stress. Finally, rewiring of the Hsp70‐translational machinery interactome during heat stress hinted at a mechanism that involves interactions with nascent peptides and the exit tunnel. Our findings suggest that regulation of translation elongation in general, and by chaperones in particular, represents a major component of the cellular stress response.

Widespread Regulation of Translation by Elongation Pausing in Heat Shock

Global repression of protein synthesis is a hallmark of the cellular stress response and has been attributed primarily to inhibition of translation initiation, although this mechanism may not always explain the full extent of repression. Here, using ribosome footprinting, we show that 2 hr of severe heat stress triggers global pausing of translation elongation at around codon 65 on most mRNAs in both mouse and human cells. The genome-wide nature of the phenomenon, its location, and features of protein N termini suggested the involvement of ribosome-associated chaperones. After severe heat shock, Hsp70's interactions with the translational machinery were markedly altered and its association with ribosomes was reduced. Pretreatment with mild heat stress or overexpression of Hsp70 protected cells from heat shock-induced elongation pausing, while inhibition of Hsp70 activity triggered elongation pausing without heat stress. Our findings suggest that regulation of translation elongation in general, and by chaperones in particular, represents a major component of cellular stress responses.

Mechanism and regulation of translation elongation

The mechanism of elongation during protein synthesis has many similarities between bacteria and eukaryotes. However, many unique aspects of elongation are found in eukaryotic systems, including some that can be capitalized on to treat infectious diseases. The eukaryotic elongation factor 1A (eEF1A) binds and bundles actin, which when altered affects gene expression at the level of initiation of translation. eEF2 is subject to ADP ribosylation by bacterial toxins from Corynebacterium diphtheria and Pseudomonas aeruginosa. This leads to inhibition of its function in translation elongation. A novel Saccharomyces cerevisiae system has been established to directly address the effects of ADP‐ribosylation on eEF2's role in translation elongation in vivo. This system employs eEF2 mutations identified within the anticodon mimicry domain which display dominant resistance to galactose‐induced diphtheria toxin and also help demonstrate the key interactions required for translocation. eEF3 is a fungal specific proteins required for elongation in vitro and in vivo. The interaction of this protein with the ribosome appears to be linked to the activity of Stm1p. The nature of this interaction, and the role of a chromodomain like insertion in the second ATP binding domain of eEF3, likely play key roles in the mechanism of action of this protein.

A new view of protein synthesis: Mapping the free energy landscape of the ribosome using single‐molecule FRET

This article reviews the application of single-molecule fluorescence resonance energy transfer (smFRET) methods to the study of protein synthesis catalyzed by the ribosome. smFRET is a powerful new technique that can be used to investigate dynamic processes within enzymes spanning many orders of magnitude. The application of wide-field smFRET imaging methods to the study of dynamic processes in the ribosome offers a new perspective on the mechanism of protein synthesis. Using this technique, the structural and kinetic parameters of tRNA motions within wild-type and specifically mutated ribosome complexes have been obtained that provide valuable new insights into the mechanism and regulation of translation elongation. The results of these studies are discussed in the context of current knowledge of the ribosome mechanism from both structural and biophysical perspectives.

Regulation of translation elongation by guanine nucleotide exchange factor, eEF1Bα

Translation elongation requires the GTP-dependent activity of eEF1A. eEF1Bα functions as the catalytic subunit of the guanine nucleotide exchange factor complex of eEF1A to maintain the GTP-bound form and allow ternary complex (eEF1A-GTP-aminoacyl tRNA) formation. Structural analysis shows that domain II of eEF1A forms a hydrophobic pocket that binds eEF1Bα and is proposed to overlap the aa-tRNA binding site. Since F163 of eEF1Bα is nested in this pocket, it may stabilize eEF1A binding and channeling of aa-tRNA to eEF1A. The F163A mutant exhibits growth and translation defects. We hypothesized that W130A eEF1Bα mutation increases flexibility to compensate for the F163A defect causing intragenic suppression. W130A/F163A double mutant partially restores F163A defects in vivo. Stopped-flow kinetic analysis demonstrates that F163A eEF1Bα decreases GDP exchange 30 fold and has a higher Kd for eEF1A of 26 μM compared to 4 μM for WT. The W130A mutant increases koff of the GDP:eEF1A complex 2 fold. The W130A/F163A mutant partially suppresses F163A exchange activity but does not compensate for its eEF1A binding defect. This data suggests that intragenic suppression is due to the rate at which eEF1Bα accelerates GDP release. Further studies will utilize these eEF1Bα mutations to understand the interplay between eEF1A:eEF1Bα complex formation and the formation of the aa-tRNA:eEF1A:GTP ternary complex. Supported by NIH.

Regulation of translation elongation and phosphorylation of eEF2 in rat pancreatic acini

Regulation of translation elongation and phosphorylation of eEF2 in rat pancreatic acini

Regulation of translation elongation and phosphorylation of eEF2 in rat pancreatic acini

While pancreatic protein synthesis and the initiation of translation are regulated by hormones and neurotransmiters, whether the elongation process is also regulated is unknown. Stimulatory doses of cholecystokinin (CCK) (100 pM), bombesin (10 nM), and carbachol (10 μM) increased elongation rates (measured as ribosomal half-transit time) in pancreatic acini in vitro. At the same time these secretagogues reduced elongation factor 2 (eEF2) phosphorylation, the main factor known to regulate elongation, and increased the phosphorylation of the eEF2 kinase. The mTOR inhibitor rapamycin reversed the dephosphorylation of eEF2 induced by CCK, as did treatment with the p38 MAPK inhibitor SB202190, the MEK inhibitor PD98059, and the phosphatase inhibitor calyculin A. Neither rapamycin, SB202190, PD98059 nor calyculin A had an effect on CCK mediated eEF2 kinase phosphorylation. Translation elongation in pancreatic acinar cells is likely regulated by eEF2 through the mTOR, p38, and MEK pathways, and modulated through PP2A.

Regulation of translation elongation in cyanobacteria: membrane targeting of the ribosome nascent-chain complexes controls the synthesis of D1 protein.

The photosystem II reaction centre protein D1 is encoded by the psbA gene. The D1 protein is stable in darkness but undergoes rapid turnover in the light. Here, we show that, in cyanobacterium Synechocystis sp. PCC6803, the synthesis of the D1 protein is regulated at the level of translation elongation in addition to the previously known transcriptional regulation. When Synechocystis sp. PCC6803 cells were transferred from light to darkness, the psbA mRNA remained abundant for hours. Cytosolic ribosomes were attached to psbA transcripts in the dark, and translation continued up to a distinct pausing site. However, ribosome nascent D1 chain complexes were not targeted to the thylakoid membrane, and no full-length D1 protein was produced in darkness. The arrest in translation elongation was released in the light, concomitantly with targeting of ribosome D1 nascent-chain complexes to the thylakoid membrane, allowing the synthesis of the full-length D1 protein. Downregulation of membrane targeting of ribosome complexes was also observed in the light if damage to the D1 protein was slow. This novel type of regulation of prokaryotic translation functions to balance the synthesis and degradation of the rapidly turning over photosystem II D1 protein in Synechocystis sp. PCC6803.

Biogenesis of the chloroplast-encoded D1 protein: regulation of translation elongation, insertion, and assembly into photosystem II.

Regulation of translation elongation, membrane insertion, and assembly of the chloroplast-encoded D1 protein of photosystem II (PSII) was studied using a chloroplast translation system in organello. Translation elongation of D1 protein was found to be regulated by (1) a redox component that can be activated not only by photosynthetic electron transfer but also by reduction with DTT; (2) the trans-thylakoid proton gradient, which is absolutely required for elongation of D1 nascent chains on the thylakoid membrane; and (3) the thiol reactants N-ethylmaleimide (NEM) and iodosobenzoic acid (IBZ), which inhibit translation elongation with concomitant accumulation of distinct D1 pausing intermediates. These results demonstrate that D1 translation elongation and membrane insertion are tightly coupled and highly regulated processes in that proper insertion is a prerequisite for translation elongation of D1. Cotranslational and post-translational assembly steps of D1 into PSII reaction center and core complexes occurred independently of photosynthetic electron transfer or trans-thylakoid proton gradient but were strongly affected by the thiol reactants DTT, NEM, and IBZ. These compounds reduced the stability of the early PSII assembly intermediates, hampered the C-terminal processing of the precursor of D1, and prevented the post-translational reassociation of CP43, indicating a strong dependence of the D1 assembly steps on proper redox conditions and the formation of disulfide bonds.

Biogenesis of the Chloroplast-Encoded D1 Protein: Regulation of Translation Elongation, Insertion, and Assembly into Photosystem II

Biogenesis of the Chloroplast-Encoded D1 Protein: Regulation of Translation Elongation, Insertion, and Assembly into Photosystem II

Inventory, assembly and analysis of Bacillus subtilis ABC transport systems

We have undertaken the inventory and assembly of the ATP binding cassette (ABC) transporter systems in the complete genome of Bacillus subtilis. We combined the identification of the three protein partners that compose an ABC transporter (nucleotide-binding domain, NBD; membrane spanning domain, MSD; and solute-binding protein, SBP) with constraints on the genetic organization. This strategy allowed the identification of 86 NBDs in 78 proteins, 103 MSD proteins and 37 SBPs. The analysis of transcriptional units allows the reconstruction of 59 ABC transporters, which include at least one NBD and one MSD. A particular class of five dimeric ATPases was not associated to MSD partners and is assumed to be involved either in macrolide resistance or regulation of translation elongation. In addition, we have detected five genes encoding ATPases without any gene coding for MSD protein in their neighborhood and 11 operons that encode only the membrane and solute-binding proteins. On the bases of similarities, three ATP-binding proteins are proposed to energize ten incomplete systems, suggesting that one ATPase may be recruited by more than one transporter. Finally, we estimate that the B. subtilis genome encodes for at least 78 ABC transporters that have been split in 38 importers and 40 extruders. The ABC systems have been further classified into 11 sub-families according to the tree obtained from the NBDs and the clustering of the MSDs and the SBPs. Comparisons with Escherichia coli show that the extruders are over-represented in B. subtilis, corresponding to an expansion of the sub-families of antibiotic and drug resistance systems.